School of BioSciences - Research Publications
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ItemAndrogen and Oestrogen Affect the Expression of Long Non-Coding RNAs During Phallus Development in a Marsupial(MDPI, 2019-03)There is increasing evidence that long non-coding RNAs (lncRNAs) are important for normal reproductive development, yet very few lncRNAs have been identified in phalluses so far. Unlike eutherians, phallus development in the marsupial tammar wallaby occurs post-natally, enabling manipulation not possible in eutherians in which differentiation occurs in utero. We treated with sex steroids to determine the effects of androgen and oestrogen on lncRNA expression during phallus development. Hormonal manipulations altered the coding and non-coding gene expression profile of phalluses. We identified several predicted co-regulatory lncRNAs that appear to be co-expressed with the hormone-responsive candidate genes regulating urethral closure and phallus growth, namely IGF1, AR and ESR1. Interestingly, more than 50% of AR-associated coding genes and lncRNAs were also associated with ESR1. In addition, we identified and validated three novel co-regulatory and hormone-responsive lncRNAs: lnc-BMP5, lnc-ZBTB16 and lncRSPO4. Lnc-BMP5 was detected in the urethral epithelium of male phalluses and was downregulated by oestrogen in males. Lnc-ZBTB16 was downregulated by oestrogen treatment in male phalluses at day 50 post-partum (pp). LncRSPO4 was downregulated by adiol treatment in female phalluses but increased in male phalluses after castration. Thus, the expression pattern and hormone responsiveness of these lncRNAs suggests a physiological role in the development of the phallus.
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ItemDNA methylation dynamics in the germline of the marsupial tammar wallaby, Macropus eugenii(OXFORD UNIV PRESS, 2019-02)Parent specific-DNA methylation is the genomic imprint that induces mono-allelic gene expression dependent on parental origin. Resetting of DNA methylation in the germ line is mediated by a genome-wide re-methylation following demethylation known as epigenetic reprogramming. Most of our understanding of epigenetic reprogramming in germ cells is based on studies in mice, but little is known about this in marsupials. We examined genome-wide changes in DNA methylation levels by measuring 5-methylcytosine expression, and mRNA expression and protein localization of the key enzyme DNA methyltransferase 3 L (DNMT3L) during germ cell development of the marsupial tammar wallaby, Macropus eugenii. Our data clearly showed that the relative timing of genome-wide changes in DNA methylation was conserved between the tammar and mouse, but in the tammar it all occurred post-natally. In the female tammar, genome-wide demethylation occurred in two phases, I and II, suggesting that there is an unidentified demethylation mechanism in this species. Although the localization pattern of DNMT3L in male germ cells differed, the expression patterns of DNMT3L were broadly conserved between tammar, mouse and human. Thus, the basic mechanisms of DNA methylation-reprogramming must have been established before the marsupial-eutherian mammal divergence over 160 Mya.
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ItemTranscriptomic analysis of MAP3K1 and MAP3K4 in the developing marsupial gonad( 2018)MAPKs affect gonadal differentiation in mice and humans, but whether this applies to all mammals is as yet unknown. We investigated MAPK expression during gonadal differentiation in a distantly related mammal, the marsupial tammar wallaby, using our model of oestrogen-induced gonadal sex reversal. MAPK pathway genes in the tammar were highly conserved at the sequence and amino acid level with those of mice and humans. Marsupial MAP3K1 and MAP3K4 clustered together in a separate branch from eutherian mammals. We characterised the transcriptome of the key sex differentiation genes and those of the MAPK pathway in developing testes and also after testis culture with exogenous oestrogen. There was a marked decrease in levels of mRNA of both male-determining genes SOX9 and AMH, while levels of the female marker FOXL2 increased in male tammar gonads exposed to exogenous oestrogen. Only MAP3K1 expression increased after male gonads were cultured in media to which oestrogen was added, but the other MAPK genes were not affected by oestrogen. This study suggests that MAP3K1 can be influenced by exogenous oestrogens during gonadal differentiation in this marsupial.